The elevated concentration of alkane-oxidizing microorganisms above oil reservoirs has long been acknowledged. Their oxidation of the light hydrocarbon stream (C1-C4) of gases emitted from petroleum and gas fields is of particular interest.
These hydrocarbon-oxidizing microorganisms are found associated with the presence of petroleum-like compositions. For example, they can be found living several centimeters beneath the soil surface above oil-bearing substrata. The light hydrocarbons rising to the soil surface serve as an energy source for these aerobic microorganisms. For microorganisms that utilize hydrocarbons C2-C4 though, these hydrocarbons do not serve as an exclusive substrate. For example, some of these microorganisms can utilize polysaccharides and monosaccharides as well. Therefore, these organisms may be present in many different soils. However, the detection of a large population of short n-alkane-oxidizing microorganisms (C2-C4) in a sample indicates the existence of constant supply of these gaseous short-chain hydrocarbons and is thus a good indicator for the presence of oil accumulations.
Since the 1930's, alkane-oxidizing bacteria have been used to detect the presence of undiscovered petroleum reservoirs. Microbiologists G. A. Mogilewskii (1938) in the U.S.S.R. and M. S. Taggart (1941) and L. W. Blau (1942) in the United States all described the use of measuring concentrations of HCO microorganisms, in surface soil samples, as indicators of oil and gas fields in the deeper subsurface (Wagner et al., 2002). Wagner, M., J. Piske, and R. Smit (2002) “Case histories of microbial prospection for oil and gas, onshore and offshore in northwest Europe, in Surface exploration case histories: Applications of geochemistry, magnetics, and remote sensing,” D. Schumacher and L. A. LeSchack, eds., AAPG Studies in Geology No. 48 and SEG Geophysical References Series No. 11: 453-479. Traditional methods for detecting these microorganisms employ a combination of cell culturing and counting, requiring days or weeks to complete. In addition, more fastidious organisms may not grow in the artificial media provided so counts will be an underestimation of actual cell numbers.
PCR can provide a rapid, specific and reliable method of detecting bacteria and other microorganisms within a matter of hours. While PCR primers for the detection of monooxygenases, including methane monooxygenase found in methanotrophs, have been reported in literature (see, for example Stienkamp et al., (2001) Current Microbiology Vol. 42:316-322 and Baldwin et al. (2003) Applied and Environmental Microbiology, p. 3350-3358; McDonald, et al. (1995) “Appl. Environ. Microbiol. 61:116-121), the use of PCR and primers in the detection of propane monooxygenase and butane monooxygenase is novel.
Although the detection of methanotrophs in a soil sample may indicate a source of subsurface methane gas, it cannot conclusively elucidate its origins. See, for example, Brisbane et al., (1965) Annual Rev. Microbiol. 19:351-364). Methane gas can originate from both biogenic and non-biogenic sources. Methanogenic bacteria are a major source of biogenic methane when subsurface conditions are anaerobic in the presence of large amounts of organic material (e.g. swamps and landfills). Conversely, underground sources of propane and butane derive exclusively from deposits of gas or crude oil. For this reason, the detection of propane monooxygenase or butane monooxygenase in a given soil sample can be used as an accurate method to detect a previously undiscovered cache of crude petroleum.